We are interested in the causal relationships that tie repeated fluctuations in oxygen to intravitreous neovascularization (IVNV) in retinopathy of prematurity (ROP) and vascularization of previously avascular retina (VPAR). Our overall hypothesis is that repeated fluctuations in oxygen cause dysregulation of the vascular endothelial growth factor (VEGF) isoforms and receptors within regions of the developing retina creating a microenvironment that promotes unwanted IVNV and later blindness. We propose that early in retinal vascular development, fluctuations in oxygen result in an angiogenic inhibitory effect due, in part, to a disturbed balance in the regulation of the angiogenic agonist, VEGF, and angiogenic inhibitory factor, pigment epithelium-derived factor (PEDF). Later, repeated oxygen fluctuations cause oxidative injury, cell apoptosis, and signaling through pathways downstream of the gp130 receptor subunit/transducing protein causing further dysregulation of VEGF isoforms (especially the pathologic VEGF164). All of these events promote IVNV. We will use the established "50/10 OIR" model (newborn rats reared under conditions of controlled 24-hour oxygen fluctuations and return to room air). Specifically we will determine whether 1a) specific blocking of the predominant pathological isoform, VEGF164, with antibody will reduce IVNV and promote VPAR, lb) VEGF and PEDF are altered leading to an angiogenic inhibitory effect at early time points (e.g., increased PEDF and/or decreased VEGF) resulting in delayed retinal vascularization, and that, at later time points, during IVNV, the balance will shift so that angiogenic stimulation occurs (e.g., increased VEGF and/or reduced PEDF), 2a) increased reactive oxygen species (ROS) occur at the junction of vascular and avascular retina and cause endothelial cell apoptosis, maintaining peripheral avascularity and interfering with VPAR, and ROS will increase expression of VEGF164 and VEGFR2, promoting IVNV, 3a) activation of gp130 will activate JAK/STAT pathways to increase expression of VEGF164, VEGFR2, and promote IVNV, and 3b) interleukin-6 (IL-6) will reduce endothelial cell apoptosis, IVNV, and promote VPAR, whereas leukemia inhibitory factor (LIF) will increase endothelial cell apoptosis and IVNV. Methods will include RT-PCR to measure growth factor (VEGF isoforms, PEDF) and receptor (VEGFR1 and VEGFR2) mRNAs; ELISA and Western blot to measure protein of growth factors (VEGF) and phosphorylated receptors or proteins (LIFR, gp130, IL-6R, STATs, JAK2, SOCS); intraocular injections of LIF, IL-6, or antibodies (VEGF164, VEGFR2); systemic injections of antioxidants (n-acetylcysteine) or a JAK2 inhibitor (AG490); measurement of lipid peroxides and superoxide damage (dihydroethidium); apoptosis (activated caspase-3) and confocal microscopy; and quantification of avascular and vascular retina and clock hours of IVNV. Understanding these mechanisms that cause pathologic VEGF164 expression will provide potential strategies to prevent IVNV and to facilitate regression of IVNV and promote VPAR.